1. Field of the Invention
The present invention relates generally to the genetic manipulation of a parasite genome (such as Plasmodium falciparum) through the use of a piggyBac transposable element construct, as well as to piggyBac transposable element constructs themselves, and for applications to identify, characterize, and/or create therapies protective in people against malaria. The field of the invention also relates to the field of malaria, and methods for controlling malarial-transmitting organisms such as Plasmodium falciparum through the use of the herein described piggyBac constructs.
2. Related Art
Malaria is a deadly infectious disease annually causing clinical illness in 400-600 million people, and killing millions.5 Caused by several different Plasmodium species, malaria remains endemic in many tropical and temperate climates. Traditional measures to control malaria are becoming increasingly ineffective due to widespread resistance against many of the available antimalarial drugs and insecticide resistance in the mosquito vectors of the parasite.6-9 There is an urgent need for the development of new drugs and vaccines to reverse a progressive resurgence in malaria morbidity and mortality. Better understanding of the malaria parasite biology is essential for the development of new intervention therapies and their efficient use for long-lasting control of this insidious disease.
Application of new technologies has produced a wealth of information in recent years about the genomes, proteomes, and other aspects of the basic composition of the malaria parasites. Many aspects of the parasite's biology can be inferred through these approaches, and yet the ability to utilize this new information to reveal the complex biology of Plasmodium has been slow due, at least in part, to the lack of robust and user-friendly molecular genetic tools. Manipulating the Plasmodium genome has been a great challenge due to the very low efficiency of transfection of this parasite, estimated to be about 10−6.10 Gene-targeting to identify gene functions is a cumbersome process hindered by the need to build individual targeting plasmids for each homologous recombination and a lengthy selection process for obtaining genome integrants.1-4 Further complicating this process in P. falciparum is the tendency of the parasite to maintain extra-cellular plasmid DNA as stable self-replicating episomal concatamers.11 
Transposable elements have been widely used as tools to manipulate genomes ranging from different microbes to higher invertebrates, like Drosophila, and even plants. Transposable elements do not occur naturally in many lower eukaryotes, including Plasmodium.12 Therefore, conditions needed for transposition might be harder to achieve in this parasite. So far, efficient transposon-mediated random mutagenesis in parasitic protozoa has been reported only in Leishmania.13 There has been a report of transposition in Plasmodium using the Drosophila mariner transposable element, but the transposition events occurred at a very low frequency, without the presence of the transposase, and with only two integrations in the same locus.14 
The piggyBac transposable element is derived from the cabbage looper moth Trichoplusia ni and is a member of the TTAA-target site-specific class of transposable elements.15-18 piggyBac is a Class II transposable element that exclusively targets the tetra nucleotide target site, TTAA, and always inserts and excises in a precise manner. piggyBac-based transposon vectors have been widely used to manipulate genomes of various invertebrate species, and piggyBac is currently the preferred vector of choice for enhancer trapping, gene discovery and identifying gene function in Drosophila and other insects.19-23 The attribute of piggyBac to non-preferentially integrate into the genome of Drosophila has made it more attractive than the P-element, which seems to have preferential hot spots for insertion in 5′ regulatory sequences.19 
A need continues to exist in the art of malarial disease control and molecular biology for a more complete functional analysis of the Plasmodium falciparum genome. However, this work continues to be restricted by the limited ability to effectively and efficiently genetically manipulate this malarial parasite using existing techniques and molecular tools.